The present invention relates to the use of cortisol-21-mesylate and dexamethasone-21-mesylate as inhibitors of glucocorticoid action. More particularly, the present invention relates to the use of cortisol-21-mesylate and dexamethasone-21-mesylate as irreversible anti-glucocorticoids.
A major obstacle in elucidating the mechanism of glucocorticoid steroid hormone action has been the characterization and purification of the steroid receptor. Steroid-free receptors are labile proteins which are found in very low concentrations in the cytoplasm of responsive cells. The only apparent activity of the free receptor is its ability to bind steroid reversibly. Thus the availability of a stabilized, covalent receptor-steroid complex would greatly facilitate chemical and biochemical studies of the receptor and its steroid binding site. This in turn could lead to applications in non-operable, hyper-glucocorticoid syndromes, in animals and in man.
Very few anti-glucocorticoids containing the basic glucocorticoid-specific steroid structure are known. It has now been found, in accordance with the present invention, that cortisol-21-mesylate (CM) and dexamethasone-21-mesylate, (DM) which contain both the basic glucocorticoid structure and a reactive functional group capable of yielding a covalent receptor-steroid complex, function as irreversible anti-glucocorticoids. For example, these compounds have a low but significant cell-free affinity for the glucocorticoid receptors of rat hepatoma tissue culture (HTC) cells. While occupancy of the glucocorticoid receptors in whole HTC cells is known to correlate with the induction of the enzyme tyrosine aminotransferase (TAT), CM does not induce TAT in HTC cells. DM gives little induction of TAT in HTC cells (.about.25% of full induction). However, CM and DM are potent inhibitors of TAT induction by dexamethasone in whole cells; and, the blockage of dexamethasone induction of TAT by pre-incubating HTC cells with CM or DM cannot be reversed by washing the cells. This is in contrast with the ready reversal of the effects of pre-incubating HTC cells with progesterone, which is a known, reversible anti-glucocorticoid with a non-glucocorticoid structure.
The irreversible block of TAT induction by CM or DM in whole cells is not due to cell toxicity, as shown by protein content, cell number and the ability of the cells to exclude trypan blue. In cell-free systems, pre-incubation of steroid-free receptors with CM or DM inhibits the subsequent ability of the bound receptors to exchange-bind added .sup.3 H-dexamethasone. Together, these results indicate that CM and DM are irreversible inhibitors of glucocorticoid action in whole and broken HTC cells. As such, the glucocorticoid-like CM and DM appear to be the first irreversible anti-glucocorticoids, possibly due to the formation of irreversible, covalent receptor-steroid complexes with these compounds.
The compounds employed in the present invention are .alpha.-keto-methanesulfonate derivatives of active glucocorticoid steroids. thus, cortisol-21-mesylate or CM is 4-pregnene-11.beta., 17.alpha., 21-triol-3, 20-dione-21-methanesulfonate. Dexamethasone-21-mesylate or DM is 16.alpha.-methyl-9.alpha.-fluoro-1,4-pregnadiene-11.beta., 17.alpha., 21-triol-3, 20-dione-21-methane-sulfonate. As described in detail hereinafter, these .alpha.-keto-mesylates react both in cell-free and whole cell environments, probably with the glucocorticoid receptor itself, to cause a blockage of a normal glucocorticoid induced response, i.e., the induction of the enzyme tyrosine aminotransferase. The amount of CM or DM which is effective to provide activity as an irreversible anti-glucocorticoid is in the range of from about 10.sup.-9 M to about 10.sup.-5 M.
The use of CM or DM as an irreversible anti-glucocorticoid provides for blocking of the action of glucocorticoid steroid hormones. Such blocking action would be important, for example, in the treatment of non-operable hyperglucocorticoid syndromes, such as adrenal carcinomas and ectopic ACTH syndrome, by blocking the action of elevated levels of glucocorticoids. Treatment would also be possible with patients who are hyper-responsive to glucocorticoids, such as patients with open-angle glaucoma or those who are homozygous for the postulated gene defect which causes this disease, by blocking some of the steroids and thus attenuating the responses in sensitive cells. Additional uses would include pre-operative treatment of patients with Cushing's disease in order to block the effects of glucocorticoids and thus eliminate the complications of surgery due to elevated levels of glucocorticoids. A still further use of the present method would be in studies directed to the mechanism of glucocorticoid hormone action.